Antisense RNAs control retrotransposon copy number The retrovirus-like transposon Ty1 of Saccharomyces cerevisiae is an effective model for understanding the replication of HIV, the causative agent of AIDS, and is arguably the best understood retrotransposon. Retrotransposons replicate through an RNA intermediate and have colonized most eukaryotic genomes. Studying Ty1 has also provided valuable information on human genome dynamics and evolution, and the behavior of transposon-induced mutations. RNA interference (RNAi) is a widespread mechanism used to silence infectious viruses and transposable elements, and alter gene expression, where small sequence-specific RNAs and conserved cellular proteins affect transcription, degrade mRNA transcripts or inhibit translation. Interestingly, S. cerevisiae and its closest relatives lack the genes required for RNAi, yet maintain tight control over Ty1 retrotransposition. We discovered a copy number control (CNC) system based on Ty1 antisense (AS) RNAs that inhibits retrotransposition posttranslationally. The noncoding transcripts are packaged into virus- like particles and block reverse transcription by greatly decreasing the level of the conserved Ty1 protein integrase, which is required for Ty1 as well as HIV-1 reverse transcription and integration. Our goal is to understand the mechanism of this novel form of RNAi by 1) determining how Ty1AS transcripts inhibit retrotransposition, 2) identifying sequence domains that mediate Ty1AS RNA synthesis, packaging and activity within virus-like particles, and 3) characterizing cellular genes that influence AS transcript synthesis and function. We expect this study to impact two areas of biomedical research. First, understanding Ty1 CNC can be used to help elucidate the function of HIV noncoding RNAs, improve RNA-based therapies, and identify new targets for drug development. Second, our work will inform research addressing how noncoding RNAs impact human gene expression on a broader scale, especially when one considers that over 90% of the genome is transcribed, yet the function of many of these transcripts is poorly understood. PUBLIC HEALTH RELEVANCE: Understanding the mechanism of a novel RNA-based defense against the retroviral-like transposable element Ty1 will inform areas of biomedical research concerned with elucidating the function of HIV noncoding RNAs, improving RNA-therapies to combat HIV/AIDS and identifying new targets for drug development. In addition, our work will yield insight into how noncoding RNAs impact human gene expression on a broader scale, especially when one considers that most of the genome is transcribed, yet the role many of these transcripts play in health and disease is poorly understood.